Method of inhibiting aortal smooth muscle cell proliferation using naphthyl compounds

ABSTRACT

The present invention provides a compound of formula I ##STR1## wherein R 1  is H, OH, halo, OCO(C 1  -C 6  alkyl), OCO(aryl), OSO 2  (C 4  -C 6  alkyl), OCOO(C 1  -C 6  alkyl), OCOO(aryl), OCONH (C 1  -C 6  alkyl), or OCON(C 1  -C 6  alkyl) 2  ; 
     R 2  is aryl, C 1  -C 6  alkyl, C 3  -C 6  cycloalkyl, or 4-cyclohexanol; 
     R 3  is O(CH 2 ) 2  or O(CH 2 ) 3  ; 
     R 4  and R 5  are optionally CO(CH 2 ) 3 ,CO(CH 2 ) 4 , C 1  -C 6  alkyl, or R 4  and R 5  combine to form, with the nitrogen to which they are attached, piperidine, morpholine, pyrollidine, 3-methylpyrollidine, 3,3-dimethylpyrollidine, 3,4-dimethylpyrollidine, azepine, or pipecoline; 
     R 6  is 
     
         &gt;CH(C.sub.1 -C.sub.5 alkyl), &gt;CH(C.sub.2 -C.sub.5 alkenyl), 
    
      &gt;C═CH(C 1  -C 5  alkyl), &gt;CH(aryl), &gt;C(OH)(C 1  -C 5  alkyl), &gt;C(OH)(C 2  -C 5  alkenyl), C(OH)aryl; 
     and phamaceutically acceptable salts thereof. 
     The present invention further provides pharmaceutical compositions containing compounds of formula I, optionally containing estrogen or progestin, and the use of such compounds alone, or in combination with estrogen or progestin, for alleviating the symptoms of post-menopausal syndrome, particularly osteoporosis, cardiovascular related pathological conditions, and estrogen-dependent cancer. As used herein, the term &#34;progestin&#34; includes compounds having progestational activity such as, for example, progesterone, norethylnodrel, nongestrel, megestrol acetate, norethindrone, and the like. 
     The compounds of the present invention also are useful for inhibiting uterine fibroid disease and endometriosis in women and aortal smooth muscle cell proliferation, particularly restenosis, in humans.

This application is a division of application Ser. No. 08/402,082 filedMar. 10, 1995.

FIELD OF THE INVENTION

This invention relates to the fields of pharmaceutical and organicchemistry and provides novel naphthyl compounds which are useful for thetreatment of the various medical indications associated withpost-menopausal syndrome, and uterine fibroid disease, endometriosis,and aortal smooth muscle cell proliferation.

BACKGROUND OF THE INVENTION

"Post-menopausal syndrome" is a term used to describe variouspathological conditions which frequently affect women who have enteredinto or completed the physiological metamorphosis known as menopause.Although numerous pathologies are contemplated by the use of this term,three major effects of post-menopausal syndrome are the source of thegreatest long-term medical concern: osteoporosis, cardiovascular effectssuch as hyperlipidemia, and estrogen-dependent cancer, particularlybreast and uterine cancer.

Osteoporosis describes a group of diseases which arise from diverseetiologies, but which are characterized by the net loss of bone mass perunit volume. The consequence of this loss of bone mass and resultingbone fracture is the failure of the skeleton to provide adequatestructural support for the body. One of the most common types ofosteoporosis is that associated with menopause. Most women lose fromabout 20% to about 60% of the bone mass in the trabecular compartment ofthe bone within 3 to 6 years after the cessation of mensus. This rapidloss is generally associated with an increase of bone resorption andformation. However, the resorptive cycle is more dominant and the resultis a net loss of bone mass. Osteoporosis is a common and serious diseaseamong post-menopausal women.

There are an estimated 25 million women in the United States, alone, whoare afflicted with this disease. The results of osteoporosis arepersonally harmful and also account for a large economic loss due itschronicity and the need for extensive and long term support(hospitalization and nursing home care) from the disease sequelae. Thisis especially true in more elderly patients. Additionally, althoughosteoporosis is not generally thought of as a life threateningcondition, a 20% to 30% mortality rate is related with hip fractures inelderly women. A large percentage of this mortality rate can be directlyassociated with post-menopausal osteoporosis.

The most vulnerable tissue in the bone to the effects of post-menopausalosteoporosis is the trabecular bone. This tissue is often referred to asspongy or cancellous bone and is particularly concentrated near the endsof the bone (near the joints) and in the vertebrae of the spine. Thetrabecular tissue is characterized by small osteoid structures whichinter-connect with each other, as well as the more solid and densecortical tissue which makes up the outer surface and central shaft ofthe bone. This inter-connected network of trabeculae gives lateralsupport to the outer cortical structure and is critical to thebio-mechanical strength of the overall structure. In post-menopausalosteoporosis, it is, primarily, the net resorption and loss of thetrabeculae which leads to the failure and fracture of bone. In light ofthe loss of the trabeculae in post-menopausal women, it is notsurprising that the most common fractures are those associated withbones which are highly dependent on trabecular support, e.g., thevertebrae, the neck of the weight bearing bones such as the femur andthe fore-arm. Indeed, hip fracture, collies fractures, and vertebralcrush fractures are hall-marks of post-menopausal osteoporosis.

At this time, the only generally accepted method for treatment ofpost-menopausal osteoporosis is estrogen replacement therapy. Althoughtherapy is generally successful, patient compliance with the therapy islow primarily because estrogen treatment frequently produces undesirableside effects.

Throughout premenopausal time, most women have less incidence ofcardiovascular disease than age-matched men. Following menopause,however, the rate of cardiovascular disease in women slowly increases tomatch the rate seen in men. This loss of protection has been linked tothe loss of estrogen and, in particular, the loss of estrogen's abilityto regulate the levels of serum lipids. The nature of estrogen's abilityto regulate serum lipids is not well understood, but evidence to dateindicates that estrogen can upregulate the low density lipid (LDL)receptors in the liver to remove excess cholesterol. Additionally,estrogen appears to have some effect on the biosynthesis of cholesterol,and other beneficial effects on cardiovascular health.

It has been reported in the literature that post-menopausal women havingestrogen replacement therapy have a return of serum lipid levels toconcentrations to those of the pre-menopausal state. Thus, estrogenwould appear to be a reasonable treatment for this condition. However,the side-effects of estrogen replacement therapy are not acceptable tomany women, thus limiting the use of this therapy. An ideal therapy forthis condition would be an agent which would regulate the serum lipidlevel as does estrogen, but would be devoid of the side-effects andrisks associated with estrogen therapy.

The third major pathology associated with post-menopausal syndrome isestrogen-dependent breast cancer and, to a lesser extent,estrogen-dependent cancers of other organs, particularly the uterus.Although such neoplasms are not solely limited to a post-menopausalwomen, they are more prevalent in the older, post-menopausal population.Current chemotherapy of these cancers has relied heavily on the use ofanti-estrogen compounds such as, for example, tamoxifen. Although suchmixed agonist-antagonists have beneficial effects in the treatment ofthese cancers, and the estrogenic side-effects are tolerable in acutelife-threatening situations, they are not ideal. For example, theseagents may have stimulatory effects on certain cancer cell populationsin the uterus due to their estrogenic (agonist) properties and they may,therefore, be contraproductive in some cases. A better therapy for thetreatment of these cancers would be an agent which is an anti-estrogencompound having negligible or no estrogen agonist properties onreproductive tissues.

In response to the clear need for new pharmaceutical agents which arecapable of alleviating the symptoms of, inter alia, post-menopausalsyndrome, the present invention provides new naphthyl compounds,pharmaceutical compositions thereof, and methods of using such compoundsfor the treatment of post-menopausal syndrome and other estrogen-relatedpathological conditions such as those mentioned below.

Uterine fibrosis iuterine fibroid disease) is an old and ever presennclinical problem which goes under a variety of names, including uterinefibroid disease, uterine hypertrophy, uterine lieomyomata, myometrialhypertrophy, fibrosis uteri, and fibrotic metritis. Essentially, uterinefibrosis is a condition where there is an inappropriate deposition offibroid tissue on the wall of the uterus.

This condition is a cause of dysmenorrhea and infertility in women. Theexact cause of this condition is poorly understood but evidence suggeststhat it is an inappropriate response of fibroid tissue to estrogen. Sucha condition has been produced in rabbits by daily administrations ofestrogen for 3 months. In guinea pigs, the condition has been producedby daily administration of estrogen for four months. Further, in rats,estrogen causes similar hypertrophy.

The most common treatment of uterine fibrosis involves surgicalprocedures both costly and sometimes a source of complications such asthe formation of abdominal adhesions and infections. In some patients,initial surgery is only a temporary treatment and the fibroids regrow.In those cases a hysterectomy is performed which effectively ends thefibroids but also the reproductive life of the patient. Also,gonadotropin releasing hormone antagonists may be administered, yettheir use is tempered by the fact they can lead to osteoporosis. Thus,there exists a need for new methods for treating uterine fibrosis, andthe methods of the present invention satisfy that need.

Endomemriosis is a condition of severe dysmenorrhea, which isaccompanied by severe pain, bleeding into the endometrial masses orperitoneal cavity and often leads to infertility. The cause of thesymptoms of this condition appear to be ectopic endometrial growthswhich respond inappropriately to normal hormonal control and are locatedin inappropriate tissues. Because of the inappropriate locations forendometrial growth, the tissue seems to initiate local inflammatory-likeresponses causing macrophage infiltration and a cascade of eventsleading to initiation of the painful response. The exact etiology ofthis disease is not well understood and its treatment by hormonaltherapy is diverse, poorly defined, and marked by numerous unwanted andperhaps dangerous side effects.

One of the treatments for this disease is the use of low dose estrogento suppress endometrial growth through a negative feedback effect oncentral gonadotropin release and subsequent ovarian production ofestrogen; however, it is sometimes necessary to use continuous estrogento control the symptoms. This use of estrogen can often lead toundesirable side effects and even the risk of endometrial cancer.

Another treatment consists of continuous administration of progestinswhich induces amenorrhea and by suppressing ovarian estrogen productioncan cause regressions of the endometrial growths. The use of chronicprogestin therapy is often accompanied by the unpleasant CNS sideeffects of progestins and often leads to infertility due to suppressionof ovarian function.

A third treatment consists of the administration of weak androgens,which are effective in controlling the endometriosis; however, theyinduce severe masculinizing effects. Several of these treatments forendometriosis have also been implicated in causing a mild degree of boneloss with continued therapy. Therefore, new methods of treatingendometriosis are desirable.

Smooth aortal muscle cell proliferation plays an important role indiseases such as atherosclerosis and restenosis. Vascular restenosisafter percutaneous transluminal coronary angioplasty (PTCA) has beenshown to be a tissue response characterized by an early and late phase.The early phase occurring hours to days after PTCA is due to thrombosiswith some vasospasms while the late phase appears to be dominated byexcessive proliferation and migration of aortal smooth muscle cells. Inthis disease, the increased cell motility and colonization by suchmuscle cells and macrophages contribute significantly to thepathogenesis of the disease. The excessive proliferation and migrationof vascular aortal smooth muscle cells may be the primary mechanism tothe reocclusion of coronary arteries following PTCA, atherectomy, laserangioplasty and arterial bypass graft surgery. See "IntimalProliferation of Smooth Muscle Cells as an Explanation for RecurrentCoronary Artery Stenosis after Percutaneous Transluminal CoronaryAngioplasty," Austin et al., Journal of the American College ofCardiology, 8: 369-375 (August 1985).

Vascular restenosis remains a major long term complication followingsurgical intervention of blocked arteries by percutaneous transluminalcoronary angioplasty (PTCA), atherectomy, laser angioplasty and arterialbypass graft surgery. In about 35% of the patients who undergo PTCA,reocclusion occurs within three to six months after the procedure. Thecurrent strategies for treating vascular restenosis include mechanicalintervention by devices such as stents or pharmacologic therapiesincluding heparin, low molecular weight heparin, coumarin, aspirin, fishoil, calcium antagonist, steroids, and prostacyclin. These strategieshave failed to curb the reocclusion rate and have been ineffective forthe treatment and prevention of vascular restenosis. See "Prevention ofRestenosis after Percutaneous Transluminal Coronary Angioplasty: TheSearch for a `Magic Bullet`," Hermats et al., American Heart Journal,122: 171-187 (July 1991).

In the pathogenesis of restenosis excessive cell proliferation andmigration occurs as a result of growth factors produced by cellularconstituents in the blood and the damaged arterial vessel wall whichmediate the proliferation of smooth muscle cells in vascular restenosis.

Agents that inhibit the proliferation and/or migration of smooth aortalmuscle cells are useful in the treatment and prevention of restenosis.The present invention provides for the use of compounds as smooth aortalmuscle cell proliferation inhibitors and, thus inhibitors of restenosis.

SUMMARY OF THE INVENTION

The present invention relates to compounds of formula I ##STR2## whereinR₁ is H, OH, halo, OCO(C₁ -C₆ alkyl), OCO (aryl), OSO₂ (C₄ -C₆ alkyl),OCOO(C₁ -C₆ alkyl), OCOO(aryl), OCONH(C₁ -C₆ alkyl), or OCON(C₁ -C₆alkyl)₂ ;

R₂ is aryl, C₁ -C₆ alkyl, C₃ -C₆ cycloalkyl, or 4-cyclohexanol;

R₃ is O(CH₂)₂ or O(CH₂)₃ ;

R₄ and R₅ are optionally CO(CH₂)₃, CO(CH₂)₄, C₁ -C₆ alkyl, or R₄ and R₅combine to form, with the nitrogen to which they are attached,piperidine, morpholine, pyrollidine, 3-methylpyrollidine,3,3-dimethylpyrollidine, 3,4-dimethylpyrollidine, azepine, orpipecoline;

R₆ is

    >CH(C.sub.1 -C.sub.5 alkyl), >CH(C.sub.2 -C.sub.5 alkenyl), >C═CH(C.sub.1 -C.sub.5 alkyl), >CH(aryl), >C(OH) (C.sub.1 -C.sub.5 alkyl), >C(OH) (C.sub.2 -C.sub.5 alkenyl), >C(OH)aryl;

and pharmaceutically acceptable salts thereof.

The present invention further relates to pharmaceutical compositionscontaining compounds of formula I, optionally containing estrogen orprogestin, and the use of such compounds, alone, or in combination withestrogen or progestin, for alleviating the symptoms of post-menopausalsyndrome, particularly osteoporosis, cardiovascular related pathologicalconditions, and estrogen-dependent cancer. As used herein, the term"estrogen" includes steroidal compounds having estrogenic activity suchas, for example, 17β-estradiol, estrone, conjugated estrogen(Premarin®), equine estrogen, 17β-ethynyl estradiol, and the like. Asused herein, the term "progestin" includes compounds havingprogestational activity such as, for example, progesterone,norethylnodrel, nongestrel, megestrol acetate, norethindrone, and thelike.

The compounds of the present invention also are useful for inhibitinguterine fibroid disease and endometriosis in women and aortal smoothmuscle cell proliferation, particularly restenosis, in humans.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention includes compounds of formula I##STR3## wherein R₁ is H, OH, halo, OCO(C₁ -C₆ alkyl), OCO(aryl), OSO₂(C₄ -C₆ alkyl), OCOO(C₁ -C₆ alkyl), OCOO(aryl), OCONH(C₁ -C₆ alkyl), orOCON(C₁ -C₆ alkyl)₂ ;

R₂ is aryl, C₁ -C₆ alkyl, C₃ -C₆ cycloalkyl, or 4-cyclohexanol;

R₃ is O(CH₂)₂ or O(CH₂)₃ ;

R₄ and R₅ are optionally CO(CH₂)₃, CO(CH₂)₄, C₁ -C₆ alkyl, or R₄ and R₅combine to form, with the nitrogen to which they are attached,piperidine, morpholine, pyrollidine, 3-methylpyrollidine,3,3-dimethylpyrollidine, 3,4-dimethylpyrollidine, azepine, orpipecoline;

R₆ is

    >CH(C.sub.1 -C.sub.5 alkyl), >CH(C.sub.2 -C.sub.5 alkenyl), >C═CH(C.sub.1 -C.sub.5 alkyl), >CH(aryl), >C(OH) (C.sub.1 -C.sub.5 alkyl), >C(OH) (C.sub.2 -C.sub.5 alkenyl), >C(OH)aryl;

and phamaceutically acceptable salts thereof.

General terms used in the description of compounds herein described beartheir usual meanings. For example, "alkyl" refers to straight orbranched aliphatic chains of 2 to 6 carbon atoms including ethyl,propyl, isopropyl, butyl, n-butyl, pentyl, isopentyl, hexyl, isohexyl,and the like. Similarly, the term "C₂ -C₆ alkene" represents straight orbranched alkenes having 2 to 6 carbons and includes propylene, ethylene,isopropylene, butylene, n-butylene, hexylene, pentylene, and the like.

The term "aryl" includes phenyl optionally substituted 1 to 3 times withC₁ -C₆ alkyl, C₁ -C₆ alkoxy, halo, amino, nitro, or hydroxy.

Naphthyl Preparation

The starting material for a route of preparing compounds of the presentinvention, are made essentially as described in U.S. Pat. No. 4,230,862,issued Oct. 28, 1980, which is herein incorporated by reference.

In general, a readily available tetralone, or a salt thereof, of theformula ##STR4## wherein R₇ is a hydroxy-protecting group, is reactedwith an acylating agent such as a phenyl benzoate of the formula##STR5## wherein R₃, R₄ and R₅ are as defined above. The reactiongenerally is carried out in the presence of a moderately strong basesuch as sodium amide and is run at ambient temperature or below.Preferred hydroxy protecting groups are C₁ -C₄ alkyl, and methyl isespecially preferred. See, e.g., the above-incorporated United Statespatents, J. W. Barton, "Protective Groups in Organic Chemistry", J. G.W. McOmie (ed.), Plenum Press, New York, N.Y., 1973, Chapter 2, and T.W. Green, "Protective Groups in Organic Synthesis", John Wiley and Sons,New York, N.Y., 1981, Chapter 7.

For the next step, the product is converted to an enol phosphatederivative generated in situ by reaction of the enol with a phosphonateprecursor such as ClP(O)(OPh)₂ and the appropriate base. Subsequentreaction with a Grignard reagent (R² MgBr) or cuprate reagent (R₂ ²CuLi), or other nucleophilic carbon species, provides compounds offormula III, below, some of which are known in the art (see, e.g. U.S.Pat. No. 4,230,862, supra): ##STR6## wherein R₂, R₃, R₄, R₅ and R₇ areas defined above, or a pharmaceutically acceptable salt thereof.Thereafter, compound III is subjected to a selective dehydrogenation ofthe 3,4-dihydro. This may be accomplished by treatment with2,3-dichloro-5,6-dycyan-1,4-benzoquinone (DDQ) at a temperature of fromabout 50° C. to about 100° C. This results in a compound of formula II.##STR7##

Compounds of formula II represent the starting material for one processfor preparing the pharmaceutically active compounds of formula I. Theα-carbon (carboxy) may then be modified to the groups defined by R₆ whenR₆ is

    >C(OH) (C.sub.1 -C.sub.5 alkyl), >C(OH) (C.sub.2 -C.sub.5 alkenyl) or >C(OH) aryl

A group such as RLi, RMgX, or other nucteophilic species at the carbonwherein R is C₁ -C₅ alkyl, C₂ -C₅ alkenyl, or aryl, is added to aformula II compound in an appropriate solvent as defined previously, ata temperature of 0° to -85° C. After a sufficient amount of time isafforded (15 minutes to 20 hours) to allow the reaction to complete,saturated aqueous sodium bicarbonate is added, and the mixture isextracted, and the combined extracts washed, dried, filtered,concentrated and purified, to produce a compound of formula Ia.

For groups where R₆ is

    >CH(C.sub.1 -C.sub.5 alkyl), >CH(C.sub.2 -C.sub.5 alkenyl), or >CH(aryl),

the α-hydroxy group is reduced from the above compound Ia with areducing agent, such as triethylsilane, followed by addition of an acidsuch as trifluoroacetic acid. After a sufficient time (15 min. to 24hours), the reaction is quenched, such as by use of an ethylacetate/saturated aqueous sodium bicarbonate mixture. The mixture isextracted, and the organics washed, dried, filtered, concentrated andpurified, to produce a compound of formula Ib. Alternative methods foraccomplishing this include (a) trialkylsilane with a Lewis acid such asEt₂ AlCl₂ or BF₃.Et₂ O (boron trifluoride etherate); (b) hydrogen olysis(H₂) with a catalyst such as palladium on carbon. In addition,dichlorodimethyl silane followed by sodium iodide is effective.

When R₆ is a group of the formula

    >C═CH(C.sub.1 -C.sub.5 -alkyl),

an appropriate compound of formula Ia in an appropriate solvent iscooled to 10° C. to -25° C. Thereafter, the hydroxy is eliminated. Suchelimination may be accomplished by adding a base such asdimethylaminopyridine (DMAP) followed by addition of methanesulfonylchloride, followed again by DMAP. Alternatively, the desired alkenes canbe prepared by reaction of the carbonyl compound with aphosphoniumylide, such as R₂ ⁺ P--CH(C₁ -C₅ alkyl). Otherorganophosphorous comounds, such as Ar₂ P(O)CHR or (RO)₂ P(O)CHR, can beemployed as well (Boutagy et al., Chem Reviews, 74, 87 (1974)).

Other compounds are prepared by replacing the R₁ and R₂ hydroxy groupswith a moiety of the formula --O--CO--(C₁ -C₆ alkyl), --O--CO--Ar inwhich Ar is optionally substituted phenyl, or --O--SO₂ --(C₄ -C₆ alky)via well known procedures. See, e.g., U.S. Pat. No. 4,358,593, supra.

For example, when a --O--CO(C₁ -C₆ alkyl) or --O--CO--Ar group isdesired, the dihydroxy compound of formula I is reacted with an agentsuch as acyl chloride, bromide, cyanide, or azide, or with anappropriate anhydride or mixed with anhydride. The reactions areconveniently carried out in a basic solvent such as pyridine, lutidine,quinoline or isoquinoline, or in a tertiary amine solvent such astriethylamine, tributylamine, methylpiperidine, and the like. Thereaction also may be carried out in an inert solvent such as ethylacetate, dimethylformamide, dimethylsulfoxide, dioxane, dimethoxyethane,acetonitrile, acetone, methyl ethyl ketone, and the like, to which atleast one equivalent of an acid scavenger, such as a tertiary amine, hasbeen added. If desired, acylation catalysts such as4-dimethylaminopyridine or 4-pyrrolidinopyridine may be used. See, e.g.,Haslam, et al., Tetrahedron, 36:2409-2433 (1980).

The acylation reactions which provide the aforementioned R₁ and R₂groups are carried out at moderate temperatures in the range from about-25° C. to about 100° C., frequently under an inert atmosphere such asnitrogen gas. However, ambient temperature is usually adequate for thereaction to run.

Such acylations of the hydroxy group also may be performed byacid-catalyzed reactions of the appropriate carboxylic acids in inertorganic solvents or heat. Acid catalysts such as sulfuric acid,polyphosphoric acid, methanesulfonic acid, and the like are used.

The aforementioned R₁ and R₂ groups also may be provided by forming anactive ester of the appropriate acid, such as the esters formed by suchknown reagents such as dicyclohexylcarbodiimide, acylimidazoles,nitrophenols, pentachlorophenol, N-hydroxysuccinimide, and1-hydroxybenzotriazole. See, e.g., Bull. Chem. Soc. Japan, 38:1979(1965), and Chem. Ber., 788 and 2024 (1970).

Each of the above techniques which provide --O--CO--(C₁ -C₆ alkyl) and--O--CO--Ar groups are carried out in solvents as discussed above. Thesetechniques which do not produce an acid product in the course of thereaction, of course, do not necessitate the use of an acid scavenger inthe reaction mixture.

When a formula I compound is desired in which R₁ and R₂ is --O--SO₂--(C₄ -C₆ alkyl), the formula I dihydroxy compound is reacted with, forexample, a derivative of the appropriate sulfonic acid such as asulfonyl chloride, bromide, or sulfonyl ammonium salt, as taught by Kingand Monoir, J. Am. Chem. Soc., 97:2566-2567 (1975). The dihydroxycompound also can be reacted with the appropriate sulfonic anhydride.Such reactions are carried out under conditions such as were explainedabove in the discussion of reaction with acid halides and the like.

Compounds of formula I can be prepared so that R₁ and R₂ bear differentbiological protecting groups or, preferably, are prepared so that R₁ andR₂ each bear the same biological protecting group. Preferred protectinggroups include --OCH₃, --O--CO--C(CH₃)₃, --O--CO--C₆ H₅, and --O--SO₂--(CH₂)₃ --CH₃.

Although the free-base form of formula I compounds can be used in themethods of the present invention, it is preferred to prepare and use apharmaceutically acceptable salt form. Thus, the compounds used in themethods of this invention primarily form pharmaceutically acceptableacid addition salts with a wide variety of organic and inorganic acids,and include the physiologically acceptable salts which are often used inpharmaceutical chemistry. Such salts are also part of this invention.Typical inorganic acids used to form such salts include hydrochloric,hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hyprosphoric, andthe like. Salts derived from organic acids, such as aliphatic mono anddicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoicand hydroxyalkandioic acids, aromatic acids, aliphatic and aromaticsulfonic acids, may also be used. Such pharmaceutically acceptable salesthus include acetate, phenylacetate, trifluoroacetate, acrylate,ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate,methoxybenzoate, methylbenzoate, o-acetoxybenzoate,naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate,caprylate, chloride, cinnamate, titrate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, terephthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate,ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate,xylenesulfonate, tartarate, and the like. A preferred salt is thehydrochloride salt.

The pharmaceutically acceptable acid addition salts are typically formedby reacting a compound of formula I with an equimolar or excess amountof acid. The reactants are generally combined in a mutual solvent suchas diethyl ether or ethyl acetate. The salt normally precipitates out ofsolution within about one hour to 10 days and can be isolated byfiltration or the solvent can be stripped off by conventional means.

The pharmaceutically acceptable salts generally have enhanced solubilitycharacteristics compared to the compound from which they are derived,and thus are often more amenable to formulation as liquids or emulsions.

The following examples are presented to further illustrate thepreparation of compounds of the present invention. It is not intendedthat the invention be limited in scope by reason of any of the followingexamples.

¹ H NMR and ¹³ C NMR are measured as indicated at 300 and 75respectively. ¹ H NMR chemical shifts are reported as δ values in ppmrelative to the NMR solvent employed. ¹ H NMR coupling constants arereported in Hertz (Hz) and refer to apparent multiplicities.Multiplicity is indicated as follows: s (singlet); δ (doublet), t(triplet), q (quartet); m (multiplet); comp (complex), br (broad), andapp (apparent). Column chromatography is performed according to themethod of Still (Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978,43, 2923) unless otherwise indicated with EM Science silica gel (230-400mesh ASTM). Radial chromatography is performed on a Chromatotron using1, 2, or 4 mm plates. All air and/or moisture sensitive reactions arerun under an argon or nitrogen atmosphere in rigorously dried glassware.In all cases, concentrations are performed under reduced pressure with arotary evaporator.

Preparation 1 ##STR8## To a solution of compound II (R₂ ═ArOH) with the4' and 6 hydroxy groups unprotected (1.00 g,) stirring in THF (20 mL) atroom temperature is added N,N-dimethylaminopyridine (1.00 g) followed byt-butyldimenhylsilylchloride (0.90 g). After 12 h, the reaction isdiluted with water and extracted with chloroform. The combined organicextracts are dried (sodium sulfate) and concentrated. The oil is takenup in ethyl acetate and the resulting precipitate filtered off. Thefiltrate is concentrated and purified by flash chromatography (silicagel, ethyl acetate) to give the desired product. Example 1 ##STR9##

To a solution of the product from Preparation 1, (2.04 g) stirring at-78° C. in THF (10 mL) is added MeLi (4.16 mL of a 1.4M solution indiethyl ether,) dropwise. After 15 min, the reaction is quenched withexcess saturated aqueous sodium bicarbonate and extracted with ethylacetate. The combined organic extracts are washed with brine, dried(MgSO₄), filtered, and concentrated. The resulting material is purifiedby radial chromatography (silica gel, 4 mm, 2.5:2.5:0.1:0.1 ofhexanes:ethyl acetate: triethylamine:MeOH) to give the desired product.

Example 2 ##STR10##

To a solution of the product from Example 1, (0.50 g) stirring in THF (5mL) at 0° C. is added tertrabutylammonium fluoride (1.74 mL of a 1.0Msolution in THF) . After 15 min, saturated aqueous sodium bicarbonate isadded and the resulting mixture extracted with ethyl acetate. Thecombined organic extracts are washed with brine, dried (MgSO₄),filtered, and concentrated. The resulting material is purified by radialchromatography (silica gel, 2 mm, 2.5:2.5:0.1:0.1 hexanes:ethylacetate:MeOH:trienhylamine) to give the desired product.

Example 3 ##STR11## To a solution of the product of Example 1, (1.77 g)stirring in CH₂ Cl₂ (50 mL) at 0° C. is added triethylsilane (2.36 mL,)followed by trifluoroacetic acid (4.72 mL). After 15 min, the reactionis quenched by carefully pouring the reaction mixture into an ethylacetate/saturated aqueous sodium bicarbonate mixture. The biphasicmixture is extracted with ethyl acetate and the combined organicextracts washed with brine, dried (MgSO₄), filtered, and concentrated.The resulting material is purified by radial chromatography (silica gel,2 mm, 2.5:2.5:0.1:0.1 hexanes:ethyl acetate: triethylamine:MeOH) to givethe desired product. Example 4 ##STR12## To a solution of the product ofExample 3 (0.50 g) stirring in THF (5 mL) at 0° added tetrabutylammoniumfluoride (1.78 mL of a 1.0M solution in THF). After 15 min, the reactionis quenched by addition of saturated aqueous sodium bicarbonate. Theresulting mixture is extracted with ethyl acetate and the combinedorganic extracts subsequently washed with brine, dried (MgSO₄),filtered, and concentrated. The resulting material is purified by radialchromatography (silica gel, 2 mm, 2.5:2.5:0.75:0.25 hexanes:ethylacetate:MeOH:triethylamine) to give the desired product. Example 5##STR13## To a solution of the product of Example 1 (1.70 g) stirring inCH₂ Cl₂ (20 mL) at 0° C. is added N,N-dimethylaminopyridine 461 mg)followed by methanesulfonyl chloride (0.27 mL). After 0.5 h, a secondportion of N,N-dimethylaminopyridine (461 mg) is added and the solutionallowed to warm to room temperature. After 20 h, the reaction mixture ispoured into brine and subsequently extracted with ethyl acetate. Thecombined organic extracts are washed with brine, dried (MgSO₄),filtered, and concentrated. Purification via radial chromatography(silica gel, 2 mm, 2.5:2.5:0.1:0.1 hexanes:ethylacetate:triethylamine:MeOH) gives the desired product. Example 6##STR14## To a solution product of Example 5 (0.42 g) stirring in THF (5mL) at 0° C. is added tetrabutylammonium fluoride (1.51 mL of a 1.0Msolution in THF). After 15 min, saturated aqueous sodium bicarbonate isadded to the reaction and the mixture subsequently extracted with ethylacetate. The combined organic extracts are washed with brine, dried(MgSO₄), filtered, and concentrated. The resulting material is purifiedby radial chromatography (silica gel, 2 mm, 2.5:2.5:0.70:0.30hexanes:ethyl acetate:MeOH:triethylamine) no give the desired product.Example 7 ##STR15##

To a solution of the product of Preparation 1 (1.00 g) stirring at -78°C. was added PhLi (1.6 mL of a 1.8M solution). After 15 min, saturatedaqueous sodium bicarbonate is added and the resulting mixture extractedwith ethyl acetate. The combined organic extracts are washed with brine,dried (MgSO₄), filtered, and concentrated. Purification of the crudematerial by radial chromatography (4 mm, silica gel, 2.5:2.5:0.01:0.005ethyl acetate:hexanes:triethylamine:MeOH) gives the desired product.

Example 8 ##STR16##

To a solution of the product of Example 7 (0.50 g) stirring in THF (5mL) at 0° C. is added tetrabutylammonium fluoride (1.60 mL of a 1.0Msolution in THF). After 15 min, saturated aqueous sodium bicarbonate isadded and the resulting mixture extracted with ethyl acetate. Thecombined organic extracts are washed with brine, dried (MgSO₄),filtered, and concentrated. The resulting material is purified by radialchromatography (silica gel, 2 mm, 2.5:2.5:0.1:0.1 hexanes:ethylacetate:MeOH:triethylamine) to give the desired product.

Test Procedure General Preparation Procedure

In the examples illustrating the methods, a post-menopausal model wasused in which effects of different treatments upon circulating lipidswere determined.

Seventy-five day old female Sprague Dawley rats (weight range of 200 to225g) are obtained from Charles River Laboratories (Potage, Mich.). Theanimals are either bilaterally ovariectomized (OVX) or exposed to a Shamsurgical procedure at Charles River Laboratories, and then shipped afterone week. Upon arrival, they are housed in metal hanging cages in groupsof 3 or 4 per cage and have ad libitum access to food (calcium contentapproximately 0.5%) and water for one week. Room temperature ismaintained at 22.2°±1.7° C. with a minimum relative humidity of 40%. Thephotoperiod in the room is 12 hours light and 12 hours dark.

Dosing Regimen Tissue Collection. After a one week acclimation period(therefore, two weeks post-OVX) daily dosing with test compound isinitiated. 17α-ethynyl estradiol or the test compound is given orally,unless otherwise stated, as a suspension in 1% carboxymethylcellulose ordissolved in 20% cyclodextrin. Animals were dosed daily for 4 days.Following the dosing regimen, animals are weighed and anesthetized witha ketamine: Xylazine (2:1, V:V) mixture and a blood sample is collectedby cardiac puncture. The animals are then sacrificed by asphyxiationwith CO₂, the uterus is removed through a midline incision,and a wetuterine weight is determined.

Cholesterol Analyis. Blood samples are allowed to clot at roomtemperature for 2 hours, and serum is obtained following centrifugationfor 10 minutes at 3000 rpm. Serum cholesterol is determined using aBoehringer Mannheim Diagnostics high performance cholesterol assay.Briefly the cholesterol is oxidized to cholest-4-en-3-one and hydrogenperoxide. The hydrogen peroxide is then reacted with phenol and4-aminophenazone in the presence of peroxidase to produce a p-quinoneimine dye, which is read spectrophotemetrically at 500 nm. Cholesterolconcentration is then calculated against a standard curve. The entireassay is automated using a Biomek Automated Workstation.

Uterine Eosinophil Peroxidase (EPO) Assay. Uteri are kept at 4° C. untiltime of enzymatic analysis. The uteri are then homogenized in 50 volumesof 50 mM Tris buffer (pH -8.0) containing 0.005% Triton X-100. Uponaddition of 0.01% hydrogen peroxide and 10 mM O-phenylenediamine (finalconcentrations) in Tris buffer, increase in absorbance is monitored forone minute at 450 nm. The presence of eosonophils in the uterus is anindication of estrogenic activity of a compound. The maximal velocity ofa 15 second interval is determined over the initial, linear portion ofthe reaction curve.

Source of Compound: 17α-ethynyl estradiol was obtained from SigmaChemical Co., St. Louis, Mo.

Osteoporpsis Test Procedure

Following the General Preparation Procedure, infra, the rats are treateddaily for 35 days (6 rats per treatment group) and sacrificed by carbondioxide asphyxiation on the 36th day. The 35 day mime period issufficient to allow maximal reduction in bone density, measured asdescribed herein. At the time of sacrifice, the uteri are removed,dissected free of extraneous tissue, and the fluid contents are expelledbefore determination of wet weight in order to confirm estrogendeficiency associated with complete ovariectomy. Uterine weight isroutinely reduced about 75% in response to ovariectomy. The uteri arethen placed in 10% neutral buffered formalin to allow for subsequenthistological analysis.

The right femurs are excised and digitilized x-rays generated andanalyzed by an image analysis program (NIH image) at the distalmetaphysis. The proximal aspect of the tibiae from these animals arealso scanned by quantitative computed tomography.

In accordance with the above procedures, compounds of the presentinvention and ethynyl estradiol (EE₂) in 20% hydroxypropylβ-cyclodextrin are orally administered to test animals.

In summary, ovariectomy of the test animals causes a significantreduction in femur density compared to intact, vehicle treated controls.Orally administered ethynyl estradiol (EE₂) prevented this loss, but therisk of uterine stimulation with this treatment is ever-present.

The compounds of the present invention prevent bone loss in a general,dose-dependent manner. Accordingly, the compounds of the presentinvention are useful for the treatment of post-menopausal syndrome,particularly osteoporosis.

MCF-7 Proliferation Assay

MCF-7 breast adenocarcinoma cells (ATCC HTB 22) are maintained in MEM(minimal essential medium, phenol red-free, Sigma, St. Louis, Mo.)suppllmented with 10% fetal bovine serum (FBS) (V/V), L-glutamine (2mM), sodium pyruvate (1 mM) , HEPES {(N- 2-hydroxyethyl!piperazine-N'-2-ethanesulfonic acid!10 mM}, non-essential amino acids and bovineinsulin (1 ug/mL) (maintenance medium). Ten days prior to assay, MCF-7cells are switched to maintenance medium supplemented with 10% dextrancoated charcoal stripped fetal bovine serum (DCC-FBS) assay medium) inplace of 10% FBS to deplete internal stores of steroids. MCF-7 cells areremoved from maintenance flasks using cell dissociated medium (Ca++/Mg++free HBSS (phenol red-free) supplemented with 10 mM HEPES and 2 mMEDTA). Cells are washed twice with assay medium and adjusted to 80,000cells/mL. Approximately 100 μL (8,000 cells) are added to flat-bottommicroculture wells (Costar 3596) and incubated at 37° C. in a 5% CO₂humidified incubator for 48 hours to allow for cell adherence andequilibration after transfer. Serial dilutions of drugs or DMSO as adiluent control are prepared in assay medium and 50 μL transferred totriplicate microcultures followed by 50 μL assay medium for a finalvolume of 200 μL. After an additional 48 hours at 37° C. in a 5% CO₂humidified incubator, microcultures are pulsed with tritiated thymidine(1 uCi/well) for 4 hours. Cultures are terminated by freezing at -70° C.for 24 hours followed by thawing and harvesting of microcultures using aSkatron Semiautomatic Cell Harvester. Samples are counted by liquidscintillation using a Wallac BetaPlace β counter.

DMBA-Induced Mammary Tumor Inhibition

Estrogen-dependent mammary tumors are produced in female Sprague-Dawleyrats which are purchased from Harlan Industries, Indianapolis, Ind. Atabout 55 days of age, the rats receive a single oral feeding of 20 mg of7,12-dimethylbenz a!anthracene (DMBA). About 6 weeks after DMBAadministration, the mammary glands are palpated at weekly intervals forthe appearance of tumors. Whenever one or more tumors appear, thelongest and shortest diameters of each tumor are measured with a metriccaliper, the measurements are recorded, and that animal is selected forexperimentation. An attempt is made to uniformly distribute the varioussizes of tumors in the treated and control groups such thataverage-sized tumors are equivalently distributed between test groups.Control groups and test groups for each experiment contain 5 to 9animals.

Compounds of Formula I are administered either through intraperitonealinjections in 2% acacia, or orally. Orally administered compounds areeither dissolved or suspended in 0.2 mL corn oil. Each treatment,including acacia and corn oil control treatment, is administered oncedaily to each test animal. Following the initial tumor measurement andselection of test animals, tumors are measured each week by theabove-mentioned method. The treatment and measurements of animalscontinue for 3 to 5 weeks at which time the final areas of the tumorsare determined. For each compound and control treatment, the change inthe mean tumor area is determined.

Uterine Fibrosis Test Procedures

Test 1

Between 3 and 20 women having uterine fibrosis are administered acompound of the present invention. The amount of compound administeredis from 0.1 to 1000 mg/day, and the period of administration is 3months.

The women are observed during the period of administration, and up to 3months after discontinuance of administration, for effects on uterinefibrosis.

Test 2

The same procedure is used as in Test 1, except the period ofadministration is 6 months.

Test 3

The same procedure is used as in Test 1, except the period ofadministration is 1 year.

Test 4

A. Induction of fibroid tumors in guinea pig.

Prolonged estrogen stimulation is used to induce leiomyomata in sexuallymature female guinea pigs. Animals are dosed with estradiol 3-5 timesper week by injection for 2-4 months or until tumors arise. Treatmentsconsisting of a compound of the invention or vehicle is administereddaily for 3-16 weeks and then animals are sacrificed and the uteriharvested and analyzed for tumor regression.

B. Implantation of human uterine fibroid tissue in nude mice.

Tissue from human leiomyomas are implanted into the peritoneal cavityand or uterine myometrium of sexually mature, castrated, remain, nudemice. Exogenous estrogen are supplied to induce growth of the explantedtissue. In some cases, the harvested tumor cells are cultured in vitroprior to implantation. Treatment consisting of a compound of the presentinvention or vehicle is supplied by gastric lavage on a daily basis for3-16 weeks and implants are removed and measured for growth orregression. At the time of sacrifice, the uteri is harvested to assessthe status of the organ.

Test 5

A. Tissue from human uterine fibroid tumors is harvested and maintained,in vitro, as primary nontransformed cultures. Surgical specimens arepushed through a sterile mesh or sieve, or alternately teased apart fromsurrounding tissue to produce a single cell suspension. Cells aremaintained in media containing 10% serum and antibiotic. Rates of growthin the presence and absence of estrogen are determined. Cells areassayed for their ability to produce complement component C3 and theirresponse to growth factors and growth hormone. In vitro cultures areassessed for their proliferative response following treatment withprogestins, GnRH, a compound of the present invention and vehicle.Levels of steroid hormone receptors are assessed weekly to determinewhether important cell characteristics are maintained in vitro. Tissuefrom 5-25 patients are utilized.

Activity in at least one of the above tests indicates the compounds ofthe present invention are of potential in the treatment of uterinefibrosis.

Endometriosis Test Procedure

In Tests 1 and 2, effects of 14-day and 21-day administration ofcompounds of the present invention on the growth of explantedendometrial tissue can be examined.

Test 1

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into three groups of equal numbers. The estrous cycleof all animals is monitored. On the day of proestrus, surgery isperformed on each female. Females in each group have the left uterinehorn removed, sectioned into small squares, and the squares are looselysutured at various sites adjacent to the mesenteric blood flow. Inaddition, females in Group 2 have the ovaries removed.

On the day following surgery, animals in Groups 1 and 2 receiveintraperitoneal injections of water for 14 days whereas animals in Group3 receive intraperitoneal injections of 1.0 mg of a compound of thepresent invention per kilogram of body weight for the same duration.Following 14 days of treatment, each female is sacrificed and theendometrial explants, adrenals, remaining uterus, and ovaries, whereapplicable, are removed and prepared for histological examination. Theovaries and adrenals are weighed.

Test 2

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into two equal groups. The estrous cycle of all animalsis monitored. On the day of proestrus, surgery is performed on eachfemale. Females in each group have the left uterine horn removed,sectioned into small squares, and the squares are loosely sutured atvarious sites adjacent to the mesenteric blood flow.

Approximately 50 days following surgery, animals assigned to Group 1receive intraperitoneal injections of water for 21 days whereas animalsin Group 2 receive intraperitoneal injections of 1.0 mg of a compound ofthe present invention per kilogram of body weight for the same duration.Following 21 days of treatment, each female is sacrificed and theendometrial explants and adrenals are removed and weighed. The explantsare measured as an indication of growth. Estrous cycles are monitored.

Test 3

A. Surgical induction of endometriosis

Autographs of endometrial tissue are used to induce endometriosis inrats and/or rabbits. Female animals at reproductive maturity undergobilateral oophorectomy, and estrogen is supplied exogenously thusproviding a specific and constant level or hormone. Autologousendometrial tissue is implanted in the peritnoneum of 5-150 animals andestrogen supplied to induce growth of the explanted tissue. Treatmentconsisting to a compound of the present invention is supplied by gastriclavage on a daily basis for 3-16 weeks, and implants are removed andmeasured for growth or regression. At the time of sacrifice, the intacthorn of the uterus is harvested to assess status of endometrium.

B. Implantation of human endometrial tissue in nude mice.

Tissue from human endometrial lesions is implanted into the peritoneumof sexually mature, castrated, female, nude mice. Exogenous estrogen issupplied to induce growth of the explanted tissue. In some cases, theharvested endometrial cells are cultured in vitro prior to implantation.Treatment consisting of a compound of the present invention supplied bygastric lavage on a daily basis for 3-16 weeks, and implants are removedand measured for growth or regression. At the time of sacrifice, theuteri is harvested to assess the status of the intact endometrium.

Test 4

A. Tissue from human endometrial lesions is harvested and maintained invitro as primary nontransformed cultures. Surgical specimens are pushedthrough a sterile mesh or sieve, or alternately teased apart fromsurrounding tissue to produce a single cell suspension. Cells aremaintained in media containing 10% serum and antibiotic. Rates of growthin the presence and absence of estrogen are determined. Cells areassayed for their ability to produce complement component C3 and theirresponse to growth factors and growth hormone. In vitro cultures areassessed for their proliferative response following treatment withprogestins, GnRH, a compound of the invention, and vehicle. Levels ofsteroid hormone receptors are assessed weekly to determine weatherimportant cell characteristics are maintained in vitro. Tissue from 5-25patients is utilized.

Activity in any of the above assays indicates that the compounds of thepresent invention are useful in the treatment of endometriosis.

Inhibition of Aortal Smooth Cell Proliferation/Restenosis Test Procedure

Compounds of the present invention have capacity to inhibit aortalsmooth cell proliferation. This can be demonstrated by using culturedsmooth cells derived from rabbit aorta, proliferation being determinedby the measurement of DNA synthesis. Cells are obtained by explantmethod as described in Ross, J. of Cell Bio. 50: 172 (1971). Cells areplated in 96 well microtiter plates for five days. The cultures becomeconfluent and growth arrested. The cells are then transferred toDulbecco's Modified Eagle's Medium (DMEM) containing 0.5-2% plateletpoor plasma, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg mlstreptomycin, 1 mC/ml ³ H-thymidine, 20 ng/ml platelet-derived growthfactor, and varying concentrations of the present compounds. Stocksolution of the compounds is prepared in dimethyl sulphoxide and thendiluted to appropriate concentration (0.01-30 mM) in the above assaymedium. Cells are then incubated at 37° C. for 24 hours under 5% CO₂/95% air. At the end of 24 hours, the cells are fixed in methanol. ³ Hthymidine incorporation in DNA is then determined by scintillationcounting as described in Bonin, et al., Exp. Cell Res. 181: 475-482(1989).

Inhibition of aortal smooth muscle cell proliferation by the compoundsof the present invention are further demonstrated by determining theireffects on exponentially growing cells. Smooth muscle cells from rabbitaortae are seeded in 12 well tissue culture plates in DMEM containing10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100mg/ml streptomycin. After 24 hours, the cells are attached and themedium is replaced with DMEM containing 10% serum, 2 mM L-glutamine, 100U/ml penicillin, 100 mg/ml streptomycin, and desired concentrations ofthe compounds. Cells are allowed to grow for four days. Cells aretreated with trypsin and the number of cells in each culture isdetermined by counting using a ZM-Coulter counter.

Activity in she above tests indicates that the compounds of the presentinvention are of potential in the treatment of restenosis.

The present invention also provides a method of alleviatingpost-menopausal syndrome in women which comprises the aforementionedmethod using compounds of Formula I and further comprises administeringto a woman an effective amount of estrogen or progestin. Thesetreatments are particularly useful for treating osteoporosis andlowering serum cholesterol because the patient will receive the benefitsof each pharmaceutical agent while the compounds of the presentinvention would inhibit undesirable side-effects of estrogen andprogestin. Activity of these combination treatments in any of thepost-menopausal tests, infra, indicates that the combination treatmentsare useful for alleviating the symptoms of post-menopausal symptoms inwomen.

Various forms of estrogen and progestin are commercially available.Estrogen-based agents include, for example, ethynyl estrogen (0.01-0.03mg/day), mestranol (0.05-0.15 mg/day), and conjugated estrogenichormones such as Premarin® (Wyeth-Ayerst; 0.3-2.5 mg/day).Progestin-based agents include, for example, medroxyprogesterone such asProvera® (Upjohn; 2.5-10 mg/day), norethylnodrel (1.0-10.0 mg/day), andnonethindrone (0.5-2.0 mg/day). A preferred estrogen-based compound isPremarin, and norethylnodrel and norethindrone are preferredprogestin-based agents.

The method of administration of each estrogen- and progestin-based agentis consistent with that which is known in the art. For the majority ofthe methods of the present invention, compounds of Formula I areadministered continuously, from 1 to 3 times daily. However, cyclicaltherapy may especially be useful in the treatment of endometriosis ormay be used acutely during painful attacks of the disease. In the caseof restenosis, therapy may be limited to short (1-6 months) intervalsfollowing medical procedures such as angioplasty.

As used herein, the term "effective amount" means an amount of compoundof the present invention which is capable of alleviating the systems ofthe various pathological conditions herein described. The specific doseof a compound administered according to this invention will, of course,be determined by the particular circumstances surrounding the caseincluding, for example, the compound administered, the route ofadministration, the state of being of the patient, and the pathologicalcondition being treated. A typical daily dose will contain a nontoxicdosage level of from about 5 mg to about 600 mg/day of a compound of thepresent invention. Preferred daily doses generally will be from about 15mg to about 80 mg/day.

The compounds of this invention can be administered by a variety ofroutes including oral, rectal, transdermal, subucutaneus, intravenous,intramuscular, and intranasal. These compounds preferably are formulatedprior to administration, the selection of which will be decided by theattending physician. Thus, another aspect of the present invention is apharmaceutical composition comprising an effective amount of a compoundof Formula I, or a pharmaceutically acceptable salt thereof, optionallycontaining an effective amount of estrogen or progestin, and apharmaceutically acceptable carrier, diluent, or excipient.

The total active ingredients in such formulations comprises from 0.1% to99.9% by weight of the formulation. By "pharmaceutically acceptable" itis meant the carrier, diluent, excipients and salt must be compatiblewith the other ingredients of the formulation, and not deleterious tothe recipient thereof.

Pharmaceutical formulations of the present invention can be prepared byprocedures known in the art using well known and readily availableingredients. For example, the compounds of formula I, with or without anestrogen or progestin compound, can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of excipients, diluents,and carriers that are suitable for such formulations include thefollowing: fillers and extenders such as starch, sugars, mannitol, andsilicic derivatives; binding agents such as carboxymethyl cellulose andother cellulose derivatives, alginates, gelatin, andpolyvinyl-pyrrolidone; moisturizing agents such as glycerol;disintegrating agents such as calcium carbonate and sodium bicarbonate;agents for retarding dissolution such as paraffin; resorptionaccelerators such as quaternary ammonium compounds; surface activeagents such as cetyl alcohol, glycerol monostearate; adsorptive carrierssuch as kaolin and bentonite; and lubricants such as talc, calcium andmagnesium stearate, and solid polyether glycols.

The compounds also can be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for example, by intramuscular, subcutaneousor intravenous routes. Additionally, the compounds are well suited toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activeingredient only or preferably in a particular physiological location,possibly over a period of time. The coatings, envelopes, and protectivematrices may be made, for example, from polymeric substances or waxes.

Compounds of formula I, alone or in combination with a pharmaceuticalagent of the present invention, generally will be administered in aconvenient formulation. The following formulation examples only areillustrative and are not intended to limit the scope of the presentinvention.

Formulations

In the formulations which follow, "active ingredient" means a compoundof formula I, or a salt or solvate thereof.

Formulation 1

Gelatin Capsules

Hard gelatin capsules are prepared using the following:

    ______________________________________                                        Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Active ingredient 0.1-1000                                                    Starch, NF        0-650                                                       Starch flowable powder                                                                          0-650                                                       Silicone fluid 350 centistrokes                                                                 0-15                                                        ______________________________________                                    

The formulation above may be changed in compliance with the reasonablevariations provided.

A tablet formulation is prepared using the ingredients below:

Formulation 2

Tablets

    ______________________________________                                        Ingredient       Quantity (mg/tablet)                                         ______________________________________                                        Active ingredient                                                                               2.5-1000                                                    Cellulose, microcrystalline                                                                    200-650                                                      Silicon dioxide, fumed                                                                         10-650                                                       Stearate acid    5-15                                                         ______________________________________                                    

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 2.5-1000 mg of active ingredientare made up as follows:

Formulation 3

Tablets

    ______________________________________                                        Ingredient         Quantity (mg/tablet)                                       ______________________________________                                        Active ingredient  25-1000                                                    Starch             45                                                         Cellulose, microcrystalline                                                                      35                                                         Polyvinylpyrrolidone                                                                             4                                                          (as 10% solution in water)                                                    Sodium carboxymethyl cellulose                                                                   4.5                                                        Magnesium stearate 0.5                                                        Talc               1                                                          ______________________________________                                    

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 ml dose aremade as follows:

Formulation 4

Suspensions

    ______________________________________                                        Ingredient         Quantity (mg/5 ml)                                         ______________________________________                                        Active ingredient  0.1-1000    mg                                             Sodium carboxymethyl cellulose                                                                   50          mg                                             Syrup              1.25        mg                                             Benzoic acid solution                                                                            0.10        mL                                             Flavor             q.v.                                                       Color              q.v.                                                       Purified water to  5           mL                                             ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor, and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

An aerosol solution is prepared containing the following ingredients:

Formulation 5

Aerosol

    ______________________________________                                        Ingredient         Quantity (% by weight)                                     ______________________________________                                        Active ingredient  0.25                                                       Ethanol            25.75                                                      Propellant 22 (Chlorodifluoromethane)                                                            70.00                                                      ______________________________________                                    

The active ingredient is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to 30° C., and transferred to afilling device. The required amount is then fled no a stainless steelcontainer and diluted with the remaining propellant. The valve units arethen fitted to the container.

Suppositories are prepared as follows:

Formulation 6

Suppositories

    ______________________________________                                        Ingredient       Quantity (mg/suppository)                                    ______________________________________                                        Active ingredient                                                                                250                                                        Saturated fatty acid glycerides                                                                2,000                                                        ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimal necessary heat. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

An intravenous formulation is prepared as follows:

Formulation 7

Intravenous Solution

    ______________________________________                                        Ingredient      Quantity                                                      ______________________________________                                        Active ingredient                                                                             50           mg                                               Isotonic saline 1,000        mL                                               ______________________________________                                    

The solution of the above ingredients is intravenously administered to apatient at a rate of about 1 mL per minute.

Formulation 8

Combination Capsule I

    ______________________________________                                        Ingredient    Quantity (mg/capsule)                                           ______________________________________                                        Active ingredient                                                                           50                                                              Premarin      1                                                               Avicel pH 101 50                                                              Starch 1500   117.50                                                          Silicon Oil   2                                                               Tween 80      0.50                                                            Cab-O-Sil     0.25                                                            ______________________________________                                    

Formulation 9

Combination Capsule II

    ______________________________________                                        Ingredient    Quantity (mg/capsule)                                           ______________________________________                                        Active ingredient                                                                           50                                                              Norethylnodrel                                                                              5                                                               Avicel pH 101 82.50                                                           Starch 1500   90                                                              Silicon Oil   2                                                               Tween 80      0.50                                                            ______________________________________                                    

Formulation 10

Combination Tablet

    ______________________________________                                        Ingredient     Quantity (mg/capsule)                                          ______________________________________                                        Active ingredient                                                                            50                                                             Premarin       1                                                              Corn Starch NF 50                                                             Povidone, K29-32                                                                             6                                                              Avicel pH 101  41.50                                                          Avicel pH 102  136.50                                                         Crospovidone XL10                                                                            2.50                                                           Magnesium Stearate                                                                           0.50                                                           Cab-O-Sil      0.50                                                           ______________________________________                                    

We claim:
 1. A method for inhibiting aortal smooth muscle cellproliferation comprising administering to a human in need of suchtreatment an effective amount of a compound of formula I ##STR17##wherein R₁ is H, OH, halo, OCO(C₁ -C₆ alkyl), OCO(aryl), OSO₂ (C₄ -C₆alkyl), OCOO(C₁ -C₆ alkyl), OCOO(aryl), OCONH(C₁ -C₆ alkyl), or OCON(C₁-C₆ alkyl)₂ ;R₂ is aryl, C₁ -C₆ alkyl, C₃ -C₆ cycloalky, or4-cyclohexanol; R₃ is O(CH₂)₂ or O(CH₂)₃ ; R₄ and R₅ are optionallyCO(CH₂)₂ CH₃, CO(CH₂)₃ CH₃, C₁ -C₆ alkyl, or R₄ and R₅ combine to form,with the nitogen to which they are attached, piperidine, morpholine,pyrrolidine, 3-mythlprrolidine, 3,3-dimethylpyrrolidine,3,4-dimethylpyrrolidine, azepine, or pipecoline; R₆ is

    >C═CH.sub.2 >CH(C.sub.1 -C.sub.5 alkyl), >CH(C.sub.2 -C.sub.5 alkenyl), >C═CH(C.sub.1 -C.sub.5 alkyl), >CH(aryl), >C(OH)(C.sub.1 -C.sub.5 alkyl), >C(OH)(C.sub.2 -C.sub.5 alkenyl), >C(OH)aryl

or a pharmaceutically acceptable salt thereof.